We are interested in control of time-ordered DNA systhesis in eukaryotic cells. We propose to study this problem by focussing on a single origin of DNA synthesis known to be utilized at a precise time in the DNA synthetic (S) period. A methotrexate-resistant Chinese hamster ovary cell line which we have developed contains an amplified nucleotide sequence the unit of which is 120 kilobases long and which is repeated approximately 1000 times in tandem on a single chromosome arm. This 120 kilobase repeated sequence includes the gene coding for dihyrofolate reductase. The chromosome arm on which the amplified region is located replicates in the first few hours of a nine hour S period, and therefore must contain at least one early-replicating origin of DNA synthesis per repeated sequence. Restriction digestion of genomic DNA from this cell line shows a unique series of 18-20 fragments on agarose gels which are absent in digests of DNA isolated from the methotrexate-sensitive parental cell line. In order to first locate and then isolate an early-replicating origin of DNA synthesis from the DNA of the methotrexate-resistant cell line we propose the following experiments: 1) electron microscopic examination of spreads of chromatin for actively-transcribing "Christmas tree" structures arising from the dihydrofolate reductase gene should allow us to determine how many replication bubbles (active origins of DNA synthesis) occur within each amplified unit, and their position relative to the dihydrofolate reductase gene; 2) pulse-labelling the DNA with 14C-thymidine in early S in synchronized cells will allow us to determine which of the unique restriction fragments arising from the amplified sequence becomes labelled first (contains an origin of DNA synthesis); 3) the unique restriction fragments will be transferred to nitrocellulose sheets and will be probed with radioactive DNA prepared from the origin of DNA synthesis in SV40 in order to look for sequence homologies to any presumptive genomic origins; 4) fragments will be cloned in bacterial plasmids and transfected back into animal cells; autonomously-replicating recombinants isolated in this way will be fully characterized with reference to the time of replication within the animal cell cycle, approximate boundaries of the origin of DNA synthesis within the fragment, conformation within minichromosomes, etc.